1. Field of the Invention
Hybrid DNA technology provides the opportunity to produce any amino acid sequence from naturally occurring amino acids. However, to use this ability in a useful manner has involved the overcoming of numerous obstacles, many of them associated with unknown factors involved with replication and expression in unicellular microorganism hosts. While the basic concepts of transcription of DNA to messenger RNA, splicing, and translation have seen enormous expansions in understanding of the processes involved, nevertheless the stability of messenger RNA, the rapidity with which it is translated, the stability of foreign proteins in the microorganisms, and the efficiency of processing and secretion, are still understood only at a rudimentary level.
For many polypeptides, the economies of manufacture will determine their utility. It will therefore be important that systems be developed, where the vectors, markers, regulatory signals, and host cooperate to provide for the production of the desired polypeptide in a form and at a level of total protein in which it may be readily isolated and purified. Desirably, where the natural product is processed to a mature protein, the host may afford partial or complete processing.
It is thus of vital importance to the development of useful proteins for industrial and therapeutic purposes to efficiently produce in high yields polypeptides of interest employing systems which can be readily developed, can be stably maintained and employ a host which can be grown in industrially efficient ways.
2. Description of the Prior Art
Carrell et al., Nature (1982) 298: 329-334 provide the amino acid sequence of human .alpha..sub.1 -antitrypsin, with discussion of various known antitrypsin mutants. Kurachi et al., Proc. Natl. Acad. Sci. USA (1981) 78: 6826-6830 present the entire cDNA sequence of baboon .alpha..sub.1 -antitrypsin. A partial human cDNA clone sequence is also shown. Bollen et al., DNA (1983) 2: 255-264 describe the cloning and expression in E. coli of a full cDNA coding for human .alpha..sub.1 -antitrypsin. Courtney et al., Proc. Natl. Acad. Sci. USA (1984) 81: 669-673 describe the expression of human cDNA for .alpha..sub.1 -antitrypsin in E. coli. Kawasaki et al., "The Production of Human .alpha..sub.1 -antitrypsin in Yeast and Fission Yeast" in The Molecular Biology of Yeast, Cold Spring Harbor Laboratory, 1983, describe the expression of human .alpha..sub.1 -antitrypsin in S. cerevisiae and S. pombe employing the yeast triosephosphate isomerase promoter and terminator sequences. The protein is reported to be unglycosylated, but biologically active against trypsin and elastase.